Tomato plants with improved traits

ABSTRACT

Tomato plants exhibiting fruit with increased BRIX content are provided, together with methods of producing, identifying, or selecting plants or germplasm with an increased BRIX phenotype and lacking undesirable leaf necrosis. Such plants include tomato plants comprising introgressed genomic regions conferring increased BRIX without necrosis. Compositions, including novel polymorphic markers for detecting plants comprising introgressed alleles providing increased BRIX without necrosis, are further provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. Provisional Appl. Ser. No.62/613,617, filed Jan. 4, 2018, which is incorporated herein byreference in its entirety.

INCORPORATION OF SEQUENCE LISTING

A sequence listing containing the file named “SEMB033US_ST25.txt” whichis 36 kilobytes (measured in MS-Windows®) and created on Jan. 2, 2019,and comprises 65 sequences, is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to the field of plant breeding and morespecifically to methods and compositions for producing tomato plantsexhibiting improved fruit quality without linked deleterious traits.

BACKGROUND

Fruit quality and flavor are important traits in tomato breeding,particularly for the development of commercial varieties. Although fruitquality alleles have been identified in tomato, efforts to introducethese alleles into cultivated lines have been hindered by a lack ofspecific markers linked to the alleles, as well as the presence ofdeleterious alleles genetically linked to fruit quality alleles thatlead to unacceptable plant traits such as leaf necrosis. The use ofmarker-assisted selection (MAS) in plant breeding has made it possibleto select plants based on genetic markers linked to traits of interest.However, accurate markers for identifying or tracking desirable traitsin plants are frequently unavailable even if a gene associated with thetrait has been characterized. These difficulties are further complicatedby factors such as polygenic or quantitative inheritance, epistasis, andan often incomplete understanding of the genetic background underlyingexpression of a desired phenotype. In the absence of accurate andvalidated markers for use in MAS, it may not be feasible to produce newplant lines exhibiting a certain fruit quality without unacceptablenecrosis.

SUMMARY

In a first aspect, Solanum lycopersicum a plant is provided comprising arecombinant chromosomal segment on chromosome 1, wherein saidchromosomal segment comprises an introgressed BRIX allele from S.pennellii conferring increased BRIX to the fruit of said plant comparedto the fruit of a plant not comprising said allele, and wherein saidchromosomal segment lacks a deleterious allele genetically linked tosaid BRIX allele that confers necrosis to said plant when present. Incertain embodiments, said BRIX allele is located within a chromosomalsegment flanked by marker locus M3 (SEQ ID NO:11) and marker locus M11(SEQ ID NO:51) on chromosome 1 of said plant, for example within achromosomal segment flanked by marker locus M3 (SEQ ID NO:11) and markerlocus M9 (SEQ ID NO:41) on chromosome 1 of said plant, or within achromosomal segment flanked by marker locus M4 (SEQ ID NO:16) and markerlocus M5 (SEQ ID NO:21) on chromosome 1 of said plant. In someembodiments, said recombinant chromosomal segment comprises a markerlocus selected from the group consisting of M1 (SEQ ID NO:1), M2 (SEQ IDNO:6), M3 (SEQ ID NO:11), M4 (SEQ ID NO:16), M5 (SEQ ID NO:21), M6 (SEQID NO:26), M7 (SEQ ID NO:31), M8 (SEQ ID NO:36), M9 (SEQ ID NO:41), M10(SEQ ID NO:46), M11 (SEQ ID NO:51), M12 (SEQ ID NO:56), and M13 (SEQ IDNO:61) on chromosome 1. In further embodiments, said plant comprises anS. lycopersicum allele at marker locus M2 (SEQ ID NO:6) and an S.pennellii allele at marker locus M3 (SEQ ID NO:11). In yet furtherembodiments, said plant comprises an S. pennellii allele at marker locusM9 (SEQ ID NO:41), and an S. lycopersicum allele at marker locus M10(SEQ ID NO:46). Further provided are plants comprising an S.lycopersicum allele at M2 (SEQ ID NO:6) and M10 (SEQ ID NO:46) and an S.pennellii allele at a marker locus selected from the group consisting ofM3 (SEQ ID NO:11), M4 (SEQ ID NO:16), M5 (SEQ ID NO:21), M6 (SEQ IDNO:26), M7 (SEQ ID NO:31), M8 (SEQ ID NO:36), M9 (SEQ ID NO:41), and M12(SEQ ID NO:56). In some embodiments, said BRIX allele is located withina chromosomal segment in the genome of said plant flanked by 96,871,421bp and 98,155,761 bp on the Tomato SL3.0 map. Further provided are plantparts of plant disclosed herein. In certain embodiments, saidintrogressed BRIX allele is derived from a plant of S. pennellii lineLA0716.

In another aspect, a recombinant DNA segment is provided comprising aBRIX allele that confers increased BRIX to the fruit of a plant, andlacking a deleterious allele genetically linked to said BRIX allele thatconfers necrosis to a plant. In some embodiments, said recombinant DNAsegment comprises a sequence selected from the group consisting of M1(SEQ ID NO:1), M2 (SEQ ID NO:6), M3 (SEQ ID NO:11), M4 (SEQ ID NO:16),M5 (SEQ ID NO:21), M6 (SEQ ID NO:26), M7 (SEQ ID NO:31), M8 (SEQ IDNO:36), M9 (SEQ ID NO:41), M10 (SEQ ID NO:46), M11 (SEQ ID NO:51), M12(SEQ ID NO:56), and M13 (SEQ ID NO:61). In further embodiments,recombinant DNA segments disclosed herein are comprised within a plant,plant part, plant cell, or seed, and said DNA segment may conferincreased BRIX to the fruit of said plant.

In yet another aspect, methods are provided of producing a tomato plantexhibiting fruit with increased BRIX, comprising: a) crossing the tomatoplant disclosed herein with itself or with a second tomato plant of adifferent genotype to produce one or more progeny plants; and b)selecting a progeny plant comprising said BRIX allele and lacking saiddeleterious allele. In certain embodiments, selecting said progeny plantcomprises detecting an S. Lycopersicum allele at M2 (SEQ ID NO:6) anddetecting an S. pennellii allele at M3 (SEQ ID NO:11). In furtherembodiments, selecting said progeny plant further comprises detecting anS. pennellii allele at marker locus M9 (SEQ ID NO:41), and detecting anS. lycopersicum allele at marker locus M10 (SEQ ID NO:46). In furtherembodiments, said progeny plant is an F2-F6 progeny plant. In yetfurther embodiments, producing said progeny plant comprisesbackcrossing.

In a further aspect, methods are provided of producing a tomato plantexhibiting fruit with increased BRIX, comprising introgressing into aplant a BRIX allele within a recombinant chromosomal segment flanked inthe genome of said plant by marker locus M3 (SEQ ID NO:11) and markerlocus M11 (SEQ ID NO:51) on chromosome 1, wherein said BRIX alleleconfers increased BRIX to said fruit of said plant compared to a plantnot comprising said allele, and wherein said recombinant chromosomalsegment lacks a deleterious allele genetically linked to said BRIXallele that confers increased necrosis to said plant when present. Insome embodiments, said recombinant chromosomal segment is located withina chromosomal segment flanked by marker locus M3 (SEQ ID NO:11) andmarker locus M9 (SEQ ID NO:41) on chromosome 1 of said plant, forexample within a chromosomal segment flanked by marker locus M4 (SEQ IDNO:16) and marker locus M5 (SEQ ID NO:21) on chromosome 1 of said plant.In certain embodiments, said recombinant chromosomal segment comprises amarker locus selected from the group consisting of M1 (SEQ ID NO:1), M2(SEQ ID NO:6), M3 (SEQ ID NO:11), M4 (SEQ ID NO:16), M5 (SEQ ID NO:21),M6 (SEQ ID NO:26), M7 (SEQ ID NO:31), M8 (SEQ ID NO:36), M9 (SEQ IDNO:41), M10 (SEQ ID NO:46), M11 (SEQ ID NO:51), M12 (SEQ ID NO:56), andM13 (SEQ ID NO:61). In further embodiments, said plant comprises an S.lycopersicum allele at marker locus M2 (SEQ ID NO:6) and an S. pennelliiallele at marker locus M3 (SEQ ID NO:11). In yet further embodiments,said plant comprises an S. pennellii allele at marker locus M9 (SEQ IDNO:41), and an S. lycopersicum allele at marker locus M10 (SEQ IDNO:46). In methods provided herein, introgressing may comprisebackcrossing, marker-assisted selection, or assaying said fruit of saidplant for increased BRIX. Further provided are tomato plants obtainableby the methods provided herein.

In yet a further aspect, methods are provided of selecting a tomatoplant exhibiting fruit with increased BRIX, comprising: a) crossing atomato plant provided herein with itself or with a second tomato plantof a different genotype to produce one or more progeny plants; and b)selecting a progeny plant comprising said BRIX allele and lacking saiddeleterious allele. In some embodiments, selecting said progeny plantcomprises detecting an S. Lycopersicum allele at M2 (SEQ ID NO:6) anddetecting an S. pennellii allele at M3 (SEQ ID NO:11). In furtherembodiments, selecting said progeny plant further comprises detecting anS. pennellii allele at marker locus M9 (SEQ ID NO:41), and detecting anS. lycopersicum allele at marker locus M10 (SEQ ID NO:46). In certainembodiments, said progeny plant is an F2-F6 progeny plant. In furtherembodiments, producing said progeny plant comprises backcrossing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the zone of necrosis measurement for a tomato plant.Necrosis is measured in the area 40 cm above and 40 cm below the last(youngest) fully ripened cluster of tomato fruit.

DETAILED DESCRIPTION

Fruit quality and taste are increasingly important traits in theproduction of food crops. Tomato consumers especially are interested inbetter tasting varieties. Many factors determine the flavor of a ripetomato, but one predominantly used in the industry is the soluble solidscontent, or BRIX. BRIX levels in fruit are determined by environmentalfactors and genetics. Several alleles that increase BRIX levels havebeen identified in non-cultivated plant lines, however efforts tointroduce these alleles into cultivated lines have been hindered byhorticultural deficiencies. When a breeder introgresses a trait from awild relative, observed horticultural deficiencies may be the result ofeither pleiotropy or linkage drag. Pleiotropy can typically overcome byseveral rounds of back crossing with the horticultural superiorcultivated parent. However, overcoming linkage drag is significantlymore difficult, and success is not assured.

Linkage drag is the result of unfavorable alleles tightly linked to theallele of interest. In some cases, it is possible that the unfavorablehorticultural traits are even caused by the gene of interest. Inaddition, recombination is often suppressed in regions that areintrogressed from wild relatives, especially if those relatives arefurther removed genetically. In the case of tightly linked linkage dragthe development of markers and can help to assist the breeder inovercoming the unfavorably horticultural traits. In addition,recombination events can be developed to provide breeders with thesmallest introgression of wild species DNA possible that can be usedacross breeding programs.

Efforts have been made to introgress alleles that increase the BRIXlevels of tomato from uncultivated lines into cultivated tomato lines.For example, a locus that increases the BRIX levels of tomato fruit islocated on chromosome 1 of the Solanum pennellii genome. The locus wasfound in a study of S. pennellii LA0716×S. lycopersicum cv. M82introgression lines. These lines and derived sub-lines are available onrequest from the Hebrew University of Jerusalem or the Max PlanckInstitute of Molecular Plant Physiology (Alseekh, et al. 2013). Inaddition, LA0716 can be obtained from the Tomato Genetic Resource Centrein Davis, Calif., USA. However, these introgressions of the BRIX locuson chromosome 1 also carry a closely linked deleterious allele thatcauses necrosis of the leaves. This leaf necrosis is the most apparentin low-light conditions and is thus most readily found in tomato plantsgrown during the winter months. Leaf necrosis occurs often in tomatoplants during a growth cycle, generally in the older lower leaves. Thisis can generally be tolerated by growers. However, the leaf necrosiscaused by the allele closely linked to the BRIX increasing allele fromS. pennellii on chromosome 1 occurs on the leaves around the ripeningtruss. This is an unacceptable trait for tomato growers and any varietyexhibiting this type of necrosis is immediately considered unmarketablebecause of this. Obtaining fruit with increased BRIX content withoutunacceptable necrosis therefore remains a significant problem.

The present inventors have for the first time mapped both the BRIXincreasing alleles and the linked deleterious necrosis alleles onchromosome 1, and developed a set of markers which can track thepresence or absence of the BRIX alleles and the necrosis alleles duringplant breeding. The inventors have further produced a reducedintrogression comprising the BRIX increasing QTL on chromosome 1 thatlacks the associated leaf necrosis linkage drag from S. pennellii. TheBRIX QTL has been mapped to a 6.6 cM region between marker M3 (SEQ IDNO:11; a SNP change [A/G] at 96,871,421 bp), and marker M10 (SEQ IDNO:46; a SNP change [C/T] at 98,155,761 bp), while the leaf necrosis QTLis at about 1.2 cM distant from the BRIX locus at marker M1 (SEQ IDNO:1; a SNP change [G/C] at 96,680,481 bp), the proximal end of the BRIXQTL. To uncouple the two loci, the inventors have further developednovel molecular marker, M2 (SEQ ID NO:6; a SNP change [T/G] at96,761,293 bp), that can be used to select for the increased BRIX QTLfrom S. pennellii and against the leaf necrosis QTL from S. pennellii.

The present inventors have discovered for the first time that thedeleterious necrosis alleles can be removed from a plant containing aBRIX increasing introgression on chromosome 1 by selecting plants with arecombination event between marker loci M2 (SEQ ID NO:6) and M3 (SEQ IDNO:11), where marker locus M2 (SEQ ID NO:6) provides the S. lycopersicumallele and marker locus M3 (SEQ ID NO:11) the S. pennellii allele. Inthese plants the necrosis locus and BRIX increasing locus are uncoupled.In some embodiments, the size of S. pennellii introgression can vary atthe distal end of the BRIX increasing QTL and can include the remainderof the distal arm of chromosome 1.

In further embodiments, in addition to a recombination event betweenmarker loci M2 (SEQ ID NO: 6) and M3 (SEQ ID NO:11), a furtherrecombination event between marker locus M9 (SEQ ID NO:41; a SNP change[A/G] at 97,237,436 bp), and marker locus M10 (SEQ ID NO:46) isdetected. In this embodiment, an S. pennellii allele is present atmarker locus M9 and an S. lycopersicum allele is present at M10. Thisversion removes unwanted or unnecessary S. pennellii DNA at both theproximal and distal sides of the QTL resulting in introgression of theminimum amount of S. pennellii DNA needed to achieve an increased BRIXphenotype without leaf necrosis linkage drag.

In further embodiments, in addition to a recombination event betweenmarker loci M2 (SEQ ID NO:6) and M4 (SEQ ID NO:16), a furtherrecombination event between marker locus M12 (SEQ ID NO:56; a SNP change[G/A] at 97,062,157 bp), and marker locus M13 (SEQ ID NO:61; a SNPchange [T/G] at 97,261,644 bp) is detected. In this embodiment, an S.pennellii allele is present at marker locus M12 and an S. lycopersicumallele is present at M13. This version removes unwanted or unnecessaryS. pennellii DNA at both the proximal and distal sides of the QTLresulting in introgression of the minimum amount of S. pennellii DNAneeded to achieve an increased BRIX phenotype without leaf necrosislinkage drag.

The invention further provides markers for tracking and identifying thenovel recombinant introgressions in plants during breeding. A summary ofuseful markers is provided in Table 1. For example, marker loci M2 (SEQID NO:6), M3 (SEQ ID NO:11), M4 (SEQ ID NO:16; a SNP change [C/G] at96,902,157 bp), M5 (SEQ ID NO:21; a SNP change [G/A] at 96,934,226 bp),M6 (SEQ ID NO:26; a SNP change [G/A] at 97,023,393 bp), M7 (SEQ IDNO:31; a SNP change [G/A] at 96,985,002 bp), M8 (SEQ ID NO:36; a SNPchange [T/A] at 96,998,916 bp), M12 (SEQ ID NO:56), M9 (SEQ ID NO:41),M13 (SEQ ID NO:61), and M10 (SEQ ID NO:46). In certain embodiments, theBRIX locus may be selected for by detecting plants with an S. pennelliiallele at one of M3 (SEQ ID NO:11), M4 (SEQ ID NO:16), M5 (SEQ IDNO:21), M6 (SEQ ID NO:26), M7 (SEQ ID NO:31), M8 (SEQ ID NO:36), M12(SEQ ID NO:56), and/or M9 (SEQ ID NO:41). Plants without necrosis maythen be detected by selecting in the previously selected plants forthose plants with an S. lycopersicum allele at M2 (SEQ ID NO:6).

The invention further provides a breeding event comprising a reducedrecombinant introgression from S. pennellii between marker loci M2 (SEQID NO:6) and M10 (SEQ ID NO: 46) which confers increased BRIX withoutnecrosis. This breeding event may be used to introgress BRIX allelesfrom S. pennellii into any desired tomato genotype without deleteriouslinkage drag.

In certain embodiments, tomato plants are provided comprising anintrogressed allele on chromosome 1, wherein said introgressed alleleconfers to the fruit of said plant increased BRIX compared to a plantnot comprising said allele, wherein said plant lacks a further allelegenetically linked to said introgressed allele, that confers necrosiswhen present.

In other embodiments, the invention provides plants comprising one ormore of the novel recombinant introgressions provided herein. Thesenovel introgressions provide fruit with increased BRIX, while avoidingthe necrosis previously associated with BRIX-increasing alleles. Methodsof producing the plants described herein are further provided.

Because genetically diverse plant lines can be difficult to cross, theintrogression of BRIX-increasing alleles into cultivated lines usingconventional breeding methods could require prohibitively largesegregating populations for progeny screens with an uncertain outcome.Marker-assisted selection (MAS) is therefore essential for the effectiveintrogression of BRIX alleles into elite cultivars without unacceptablenecrosis. However, previously known markers for increased BRIX havefailed to discriminate between donor DNA conferring a BRIX increase anddonor DNA conferring deleterious traits. This has been furthercomplicated by the previous inability to resolve the specific regionsassociated with increased BRIX. For the first time, the presentinvention enables effective MAS by providing improved and validatedmarkers for detecting genotypes associated with increased BRIX anddeleterious necrosis without the need to grow large populations ofplants to maturity in order to observe the phenotype.

The invention therefore further provides novel trait-linked markerswhich can be used to produce plants comprising novel recombinantintrogressions on chromosome 1 conferring increased BRIX levels asdescribed herein. In particular embodiments, the invention provides themarkers shown in Table 1. Other embodiments of the invention providemarkers M1 (SEQ ID NO:1), M2 (SEQ ID NO:6), M3 (SEQ ID NO:11), M4 (SEQID NO:16), M5 (SEQ ID NO:21), M6 (SEQ ID NO:26), M7 (SEQ ID NO:31), M8(SEQ ID NO:36), M9 (SEQ ID NO:41), M10 (SEQ ID NO:46), M11 (SEQ IDNO:51), M12 (SEQ ID NO:56), and M13 (SEQ ID NO:61) on chromosome 1,which can be used to track BRIX and necrosis alleles in tomato plantsduring breeding.

Methods of producing plants comprising the reduced recombinantintrogressions described herein are further provided. In some examples,donor DNA from a high BRIX donor parent is introgressed into acultivated plant line (the recurrent parent line). In certainembodiments, plants can be selected based on detection of recurrentparent DNA at marker loci M2 (SEQ ID NO:6) and M13 (SEQ ID NO:61) or M10(SEQ ID NO:46) and donor DNA at a marker locus selected from the groupconsisting of M3 (SEQ ID NO:11), M4 (SEQ ID NO:16), M5 (SEQ ID NO:21),M6 (SEQ ID NO:26), M7 (SEQ ID NO:31), M8 (SEQ ID NO:36), M12 (SEQ IDNO:56), and M9 (SEQ ID NO:41).

In certain embodiments, the invention provides methods of producing orselecting a tomato plant exhibiting fruit with increased BRIX withoutnecrosis comprising: a) crossing a tomato plant provided herein withitself or with a second tomato plant of a different genotype to produceone or more progeny plants; and b) selecting a progeny plant comprisinga BRIX allele and lacking a necrosis allele. In some embodiments,methods of the invention comprise selecting a progeny plant by detectingat least one polymorphism at a locus selected from the group consistingof marker locus M2 (SEQ ID NO:6), M3 (SEQ ID NO:11), M4 (SEQ ID NO:16),M5 (SEQ ID NO:21), M6 (SEQ ID NO:26), M7 (SEQ ID NO:31), M8 (SEQ IDNO:36), M9 (SEQ ID NO:41), M10 (SEQ ID NO:46), M12 (SEQ ID NO:56), andM13 (SEQ ID NO:61).

I. Genomic Regions, Alleles, and Polymorphisms Associated with BRIX andNecrosis in Tomato Plants

The invention provides novel introgressions of one or more allelesassociated with increased BRIX without detrimental necrosis in tomatoplants, together with polymorphic nucleic acids and linked markers fortracking the introgressions during plant breeding.

Tomato lines exhibiting BRIX are known in the art and may be usedtogether with the novel trait-linked markers provided herein inaccordance with certain embodiments of the invention. For example, thewild tomato accession Solanum pennellii LA0716 (available from theTomato Genetic Resource Center in Davis, Calif., USA), can be used as asource for BRIX-increasing alleles. Using the improved genetic markersand assays of the invention, Applicants were able to successfullyidentify novel reduced introgression from S. pennellii that conferincreased BRIX to the fruit of a plant with fewer deleterious traitswhen introgressed into a cultivated line. In certain embodiments, theinvention provides tomato plants comprising donor DNA between markerlocus M3 (SEQ ID NO:11) and M10 (SEQ ID NO:46) on chromosome 1.

The novel introgressions provided herein confer robust increases inBRIX, while avoiding the necrosis seen with conventional introgressions.The invention therefore represents a significant advance in the art.

II. Introgression of Genomic Regions Associated with Increased BRIX

Marker-assisted introgression involves the transfer of a chromosomalregion defined by one or more markers from a first genetic background toa second. Offspring of a cross that contain the introgressed genomicregion can be identified by the combination of markers characteristic ofthe desired introgressed genomic region from a first genetic backgroundand both linked and unlinked markers characteristic of the secondgenetic background.

The present invention provides novel accurate markers for identifyingand tracking introgression of one or more of the genomic regionsdisclosed herein from a donor plant comprising BRIX-increasing allelesinto a cultivated line. The invention further provides markers foridentifying and tracking the novel introgressions disclosed hereinduring plant breeding, including the markers set forth in Table 1.

Markers within or linked to any of the genomic intervals of the presentinvention may be useful in a variety of breeding efforts that includeintrogression of genomic regions associated with increased BRIX into adesired genetic background. For example, a marker within 40 cM, 20 cM,15 cM, 10 cM, 5 cM, 2 cM, or 1 cM of a marker associated with increasedBRIX levels described herein can be used for marker-assistedintrogression of genomic regions associated with a disease resistantphenotype.

Tomato plants comprising one or more introgressed regions associatedwith a desired phenotype wherein at least 10%, 25%, 50%, 75%, 90%, or99% of the remaining genomic sequences carry markers characteristic ofthe recurrent parent germplasm are also provided. Tomato plantscomprising an introgressed region comprising regions closely linked toor adjacent to the genomic regions and markers provided herein andassociated with an increased BRIX phenotype are also provided.

III. Development of Tomato Varieties with Increased BRIX

For most breeding objectives, commercial breeders work within germplasmthat is “cultivated,” “cultivated type,” or “elite.” These cultivatedlines may be used as recurrent parents or as a source of recurrentparent alleles during breeding. Cultivated or elite germplasm is easierto breed because it generally performs well when evaluated forhorticultural performance. Many cultivated tomato types have beendeveloped and are known in the art as being agronomically elite andappropriate for commercial cultivation. However, the performanceadvantage a cultivated germplasm provides can be offset by a lack ofallelic diversity. Breeders generally accept this tradeoff becauseprogress is faster when working with cultivated material than whenbreeding with genetically diverse sources.

In contrast, when cultivated germplasm is crossed with non-cultivatedgermplasm, a breeder can gain access to novel alleles from thenon-cultivated type. Non-cultivated germplasm may be used as a source ofdonor alleles during breeding. However, this approach generally presentssignificant difficulties due to fertility problems associated withcrosses between diverse lines, and negative linkage drag from thenon-cultivated parent. For example, non-cultivated tomato types canprovide alleles associated with increased fruit quality. However, thesenon-cultivated types may have poor horticultural qualities such asincreased necrosis.

The process of introgressing desirable genes from non-cultivated linesinto elite cultivated lines while avoiding problems with linkage drag orlow heritability is a long and often arduous process. In deployingalleles derived from wild relatives it is often desirable to introduce aminimal or truncated introgression that provides the desired trait butlacks detrimental effects. To aid introgression reliable marker assaysare preferable to phenotypic screens. Success is furthered bysimplifying genetics for key attributes to allow focus on genetic gainfor quantitative traits such as increased fruit quality. Moreover, theprocess of introgressing genomic regions from non-cultivated lines canbe greatly facilitated by the availability of accurate markers for MAS.

One of skill in the art would therefore understand that the alleles,polymorphisms, and markers provided by the invention allow the trackingand introduction of any of the genomic regions identified herein intoany genetic background. In addition, the genomic regions associated withincreased BRIX disclosed herein can be introgressed from one genotype toanother and tracked using MAS. Thus, the inventors' discovery ofaccurate markers associated with increased BRIX will facilitate thedevelopment of tomato plants having beneficial phenotypes. For example,seed can be genotyped using the markers of the present invention toselect for plants comprising desired genomic regions associated withincreased BRIX. Moreover, MAS allows identification of plants homozygousor heterozygous for a desired introgression.

Inter-species crosses can also result in suppressed recombination andplants with low fertility or fecundity. For example, suppressedrecombination has been observed for the tomato nematode resistance geneMi, the Mla and Mlg genes in barley, the Yr17 and Lr20 genes in wheat,the Run1 gene in grapevine, and the Rma gene in peanut. Meioticrecombination is essential for classical breeding because it enables thetransfer of favorable alleles across genetic backgrounds, the removal ofdeleterious genomic fragments, and pyramiding traits that aregenetically tightly linked. Therefore, in the absence of accuratemarkers, suppressed recombination forces breeders to enlarge segregatingpopulations for progeny screens in order to arrive at the desiredgenetic combination.

Phenotypic evaluation of large populations is time-consuming,resource-intensive and not reproducible in every environment.Marker-assisted selection offers a feasible alternative. Molecularassays designed to detect unique polymorphisms, such as SNPs, areversatile. However, they may fail to discriminate alleles within andamong tomato species in a single assay. Structural rearrangements ofchromosomes such as deletions impair hybridization and extension ofsynthetically labeled oligonucleotides. In the case of duplicationevents, multiple copies are amplified in a single reaction withoutdistinction. The development and validation of accurate and highlypredictive markers are therefore essential for successful MAS breedingprograms.

IV. Marker Assisted Breeding Techniques

Genetic markers that can be used in the practice of the presentinvention include, but are not limited to, restriction fragment lengthpolymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs),simple sequence repeats (SSRs), simple sequence length polymorphisms(SSLPs), single nucleotide polymorphisms (SNPs), insertion/deletionpolymorphisms (Indels), variable number tandem repeats (VNTRs), andrandom amplified polymorphic DNA (RAPD), isozymes, and other markersknown to those skilled in the art. Marker discovery and development incrop plants provides the initial framework for applications tomarker-assisted breeding activities (U.S. Patent Pub. Nos.:2005/0204780, 2005/0216545, 2005/0218305, and 2006/00504538). Theresulting “genetic map” is the representation of the relative positionof characterized loci (polymorphic nucleic acid markers or any otherlocus for which alleles can be identified) to each other.

Polymorphisms comprising as little as a single nucleotide change can beassayed in a number of ways. For example, detection can be made byelectrophoretic techniques including a single strand conformationalpolymorphism (Orita, et al. (1989) Genomics, 8(2), 271-278), denaturinggradient gel electrophoresis (Myers (1985) EPO 0273085), or cleavagefragment length polymorphisms (Life Technologies, Inc., Gathersberg,Md.), but the widespread availability of DNA sequencing often makes iteasier to simply sequence amplified products directly. Once thepolymorphic sequence difference is known, rapid assays can be designedfor progeny testing, typically involving some version of PCRamplification of specific alleles (PASA; Sommer, et al. (1992)Biotechniques 12(1), 82-87), or PCR amplification of multiple specificalleles (PAMSA; Dutton and Sommer (1991) Biotechniques, 11(6),700-7002).

Polymorphic markers serve as useful tools for assaying plants fordetermining the degree of identity of lines or varieties (U.S. Pat. No.6,207,367). These markers form the basis for determining associationswith phenotypes and can be used to drive genetic gain. In certainembodiments of methods of the invention, polymorphic nucleic acids canbe used to detect in a tomato plant a genotype associated with increasedBRIX levels, identify a tomato plant with a genotype associated withincreased BRIX levels, and to select a tomato plant with a genotypeassociated with increased BRIX levels. In certain embodiments of methodsof the invention, polymorphic nucleic acids can be used to produce atomato plant that comprises in its genome an introgressed locusassociated with increased BRIX levels. In certain embodiments of theinvention, polymorphic nucleic acids can be used to breed progeny tomatoplants comprising a locus or loci associated with increased BRIX levels.

Genetic markers may include “dominant” or “codominant” markers.“Codominant” markers reveal the presence of two or more alleles (two perdiploid individual). “Dominant” markers reveal the presence of only asingle allele. Markers are preferably inherited in codominant fashion sothat the presence of both alleles at a diploid locus, or multiplealleles in triploid or tetraploid loci, are readily detectable, and theyare free of environmental variation, i.e., their heritability is 1. Amarker genotype typically comprises two marker alleles at each locus ina diploid organism. The marker allelic composition of each locus can beeither homozygous or heterozygous. Homozygosity is a condition whereboth alleles at a locus are characterized by the same nucleotidesequence. Heterozygosity refers to different conditions of the allele ata locus.

Nucleic acid-based analyses for determining the presence or absence ofthe genetic polymorphism (i.e. for genotyping) can be used in breedingprograms for identification, selection, introgression, and the like. Awide variety of genetic markers for the analysis of geneticpolymorphisms are available and known to those of skill in the art. Theanalysis may be used to select for genes, portions of genes, QTL,alleles, or genomic regions that comprise or are linked to a geneticmarker that is linked to or associated with increased BRIX levels intomato plants.

As used herein, nucleic acid analysis methods include, but are notlimited to, PCR-based detection methods (for example, TaqMan assays),microarray methods, mass spectrometry-based methods and/or nucleic acidsequencing methods, including whole genome sequencing. In certainembodiments, the detection of polymorphic sites in a sample of DNA, RNA,or cDNA may be facilitated through the use of nucleic acid amplificationmethods. Such methods specifically increase the concentration ofpolynucleotides that span the polymorphic site, or include that site andsequences located either distal or proximal to it. Such amplifiedmolecules can be readily detected by gel electrophoresis, fluorescencedetection methods, or other means.

One method of achieving such amplification employs the polymerase chainreaction (PCR) (Mullis et al. (1986) Cold Spring Harbor Symp. Quant.Biol. 51:263-273; European Patent 50,424; European Patent 84,796;European Patent 258,017; European Patent 237,362; European Patent201,184; U.S. Pat. Nos. 4,683,202; 4,582,788; and 4,683,194), usingprimer pairs that are capable of hybridizing to the proximal sequencesthat define a polymorphism in its double-stranded form. Methods fortyping DNA based on mass spectrometry can also be used. Such methods aredisclosed in U.S. Pat. Nos. 6,613,509 and 6,503,710, and referencesfound therein.

Polymorphisms in DNA sequences can be detected or typed by a variety ofeffective methods well known in the art including, but not limited to,those disclosed in U.S. Pat. Nos. 5,468,613, 5,217,863; 5,210,015;5,876,930; 6,030,787; 6,004,744; 6,013,431; 5,595,890; 5,762,876;5,945,283; 5,468,613; 6,090,558; 5,800,944; 5,616,464; 7,312,039;7,238,476; 7,297,485; 7,282,355; 7,270,981 and 7,250,252 all of whichare incorporated herein by reference in their entirety. However, thecompositions and methods of the present invention can be used inconjunction with any polymorphism typing method to type polymorphisms ingenomic DNA samples. These genomic DNA samples used include but are notlimited to, genomic DNA isolated directly from a plant, cloned genomicDNA, or amplified genomic DNA.

For instance, polymorphisms in DNA sequences can be detected byhybridization to allele-specific oligonucleotide (ASO) probes asdisclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863. U.S. Pat. No.5,468,613 discloses allele specific oligonucleotide hybridizations wheresingle or multiple nucleotide variations in nucleic acid sequence can bedetected in nucleic acids by a process in which the sequence containingthe nucleotide variation is amplified, spotted on a membrane and treatedwith a labeled sequence-specific oligonucleotide probe.

Target nucleic acid sequence can also be detected by probe ligationmethods, for example as disclosed in U.S. Pat. No. 5,800,944 wheresequence of interest is amplified and hybridized to probes followed byligation to detect a labeled part of the probe.

Microarrays can also be used for polymorphism detection, whereinoligonucleotide probe sets are assembled in an overlapping fashion torepresent a single sequence such that a difference in the targetsequence at one point would result in partial probe hybridization(Borevitz et al., Genome Res. 13:513-523 (2003); Cui et al.,Bioinformatics 21:3852-3858 (2005). On any one microarray, it isexpected there will be a plurality of target sequences, which mayrepresent genes and/or noncoding regions wherein each target sequence isrepresented by a series of overlapping oligonucleotides, rather than bya single probe. This platform provides for high throughput screening ofa plurality of polymorphisms. Typing of target sequences bymicroarray-based methods is described in U.S. Pat. Nos. 6,799,122;6,913,879; and 6,996,476.

Other methods for detecting SNPs and Indels include single baseextension (SBE) methods. Examples of SBE methods include, but are notlimited, to those disclosed in U.S. Pat. Nos. 6,004,744; 6,013,431;5,595,890; 5,762,876; and 5,945,283.

In another method for detecting polymorphisms, SNPs and Indels can bedetected by methods disclosed in U.S. Pat. Nos. 5,210,015; 5,876,930;and 6,030,787 in which an oligonucleotide probe having a 5′ fluorescentreporter dye and a 3′ quencher dye covalently linked to the 5′ and 3′ends of the probe. When the probe is intact, the proximity of thereporter dye to the quencher dye results in the suppression of thereporter dye fluorescence, e.g. by Forster-type energy transfer. DuringPCR, forward and reverse primers hybridize to a specific sequence of thetarget DNA flanking a polymorphism while the hybridization probehybridizes to polymorphism-containing sequence within the amplified PCRproduct. In the subsequent PCR cycle DNA polymerase with 5′→3′exonuclease activity cleaves the probe and separates the reporter dyefrom the quencher dye resulting in increased fluorescence of thereporter.

In another embodiment, a locus or loci of interest can be directlysequenced using nucleic acid sequencing technologies. Methods fornucleic acid sequencing are known in the art and include technologiesprovided by 454 Life Sciences (Branford, Conn.), Agencourt Bioscience(Beverly, Mass.), Applied Biosystems (Foster City, Calif.), LI-CORBiosciences (Lincoln, Nebr.), NimbleGen Systems (Madison, Wis.),Illumina (San Diego, Calif.), and VisiGen Biotechnologies (Houston,Tex.). Such nucleic acid sequencing technologies comprise formats suchas parallel bead arrays, sequencing by ligation, capillaryelectrophoresis, electronic microchips, “biochips,” microarrays,parallel microchips, and single-molecule arrays.

V. Definitions

The following definitions are provided to better define the presentinvention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cells of tissue culture from which tomato plants canbe regenerated, plant calli, plant clumps and plant cells that areintact in plants or parts of plants such as pollen, flowers, seeds,leaves, stems, and the like.

As used herein, the term “population” means a genetically heterogeneouscollection of plants that share a common parental derivation.

As used herein, the terms “variety” and “cultivar” mean a group ofsimilar plants that by their genetic pedigrees and performance can beidentified from other varieties within the same species.

As used herein, an “allele” refers to one of two or more alternativeforms of a genomic sequence at a given locus on a chromosome.

A “quantitative trait locus” (QTL) is a chromosomal location thatencodes for at least a first allele that affects the expressivity of aphenotype.

As used herein, a “marker” means a detectable characteristic that can beused to discriminate between organisms. Examples of such characteristicsinclude, but are not limited to, genetic markers, biochemical markers,metabolites, morphological characteristics, and agronomiccharacteristics.

As used herein, the term “phenotype” means the detectablecharacteristics of a cell or organism that can be influenced by geneexpression.

As used herein, the term “genotype” means the specific allelic makeup ofa plant.

As used herein, “elite” or “cultivated” variety means any variety thathas resulted from breeding and selection for superior agronomicperformance. An “elite plant” refers to a plant belonging to an elitevariety. Numerous elite varieties are available and known to those ofskill in the art of tomato breeding. An “elite population” is anassortment of elite individuals or varieties that can be used torepresent the state of the art in terms of agronomically superiorgenotypes of a given crop species, such as tomato. Similarly, an “elitegermplasm” or elite strain of germplasm is an agronomically superiorgermplasm.

As used herein, the term “introgressed,” when used in reference to agenetic locus, refers to a genetic locus that has been introduced into anew genetic background, such as through backcrossing. Introgression of agenetic locus can be achieved through plant breeding methods and/or bymolecular genetic methods. Such molecular genetic methods include, butare not limited to, various plant transformation techniques and/ormethods that provide for homologous recombination, non-homologousrecombination, site-specific recombination, and/or genomic modificationsthat provide for locus substitution or locus conversion.

As used herein, the terms “recombinant” or “recombined” in the contextof a chromosomal segment refer to recombinant DNA sequences comprisingone or more genetic loci in a configuration in which they are not foundin nature, for example as a result of a recombination event betweenhomologous chromosomes during meiosis.

As used herein, the term “linked,” when used in the context of nucleicacid markers and/or genomic regions, means that the markers and/orgenomic regions are located on the same linkage group or chromosome suchthat they tend to segregate together at meiosis.

The term “about” is used to indicate that a value includes the standarddeviation of error for the device or method being employed to determinethe value. The use of the term “or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only orthe alternatives are mutually exclusive, although the disclosuresupports a definition that refers to only alternatives and to “and/or.”When used in conjunction with the word “comprising” or other openlanguage in the claims, the words “a” and “an” denote “one or more,”unless specifically noted. The terms “comprise,” “have” and “include”are open-ended linking verbs. Any forms or tenses of one or more ofthese verbs, such as “comprises,” “comprising,” “has,” “having,”“includes” and “including,” are also open-ended. For example, any methodthat “comprises,” “has” or “includes” one or more steps is not limitedto possessing only those one or more steps and also covers otherunlisted steps. Similarly, any plant that “comprises,” “has” or“includes” one or more traits is not limited to possessing only thoseone or more traits and covers other unlisted traits.

EXAMPLES Example 1. Measuring BRIX and Determining Necrosis Levels

BRIX, or soluble solids content, is measured routinely in the tomatoindustry and is often used as a proxy for flavor. For the BRIXmeasurement, a BRIX meter is used, which is a digital refractometercalibrated using a series of sucrose solutions. While the method ofmeasuring BRIX in a sample is standard across the industry, the harvestmethod to produce the sample is not. All harvest methods will work ifsamples are harvested and processed at the same time, and fruit samplesare picked consistently across all plants. This is very importantbecause the BRIX level is heavily influenced by environmental conditionsand fruit ripeness. For example, fruit harvested at a different time inthe year from the same plant can have significantly different levels ofBRIX. Therefore, BRIX is often expressed as a relative value compared toa control. What the control is depends on the experiment, but can be forexample a recurrent parent, a sister line without the introgression ofinterest, or a standard variety. The following harvesting method wasused in the examples described herein. All plants were harvested twice(H1 and H2). The H1 harvest is when the second truss of the last entryhas reached 80% maturity. This means that 80% of the fruit of the trusshave fully turned to the mature fruit color. For each plant the latesttruss that reached 80% or more ripe fruit is harvested. Generally, the 2fruit at the base of the truss are taken (closest to the stem). Forsmaller fruited types this can be the 4 or 6 fruits closest to the stemof the plant. The H2 harvest is at the end of the experiment where thesame harvest protocol is used as for the H1 harvest. Fruit of a singleplant are pooled and homogenized. The BRIX content is then determinedfor the homogenized sample.

Necrosis is determined in adult plants with normal fruit load. The leafnecrosis is best observed in an environment with no artificial light, asthis can cause its own form of leaf necrosis, and under generally lowerlight conditions, as typically observed during the winter months. Plantsshould be sown such that the adult plants are in the experimental lowlight conditions from early autumn (mid-September or October) to April.Plants are grown using methods commonly used by tomato growers. Scoringof leaf necrosis starts when the positive control plants show leafnecrosis of an intermediate level, which corresponds to clear and/orintense necrosis spots on <10% of the leaf surface. Other categories forscoring can include (with increasing severity): no symptoms, minornecrosis symptoms on the leaf edge, clear necrosis symptoms on the leafedge, minor necrosis spots on <10% of the leaf surface, clear necrosisspots on <10% of the leaf surface, intense necrosis spots on <10% of theleaf surface, necrosis on <20% of the leaf surface, necrosis on <50% ofthe leaf surface, and necrosis on >50% of the leaf surface. This shouldnot be before a plant has produced two fully ripe fruit clusters. Thelevel of necrosis is scored for the leaves in a zone extending about 40cm above and about 40 cm below the truss that is ripening at that time(FIG. 1). This zone is a relative location and the exact distance fromthe ground can vary due to variation in fruit setting and growth ratesbetween the tested plants. It is important that the experiment containsadequate positive and negative control plants. Positive control plantsare for example the inbred line IL1-4 obtainable from Zamir or Fernie(Alsheekh, et al. 2013) or plants with S. pennellii alleles at marker M1(SEQ ID NO:1) and M2 (SEQ ID NO:6). A good negative control is theparent line with the S. pennellii introgression on chromosome 1. Whilethe intensity of necrosis varies depending on the genetic background,the necrosis can be observed in all cases.

Example 2. Removing Deleterious Traits on Chromosome 1

Previous research has identified several loci, including pen1 (derivedfrom S. pennellii line LA0716), with potential to increase flavor byincreasing BRIX (soluble solids content in aqueous solution (g/100 mL)).It was found that the original introgressions of this BRIX locus showeda significant increase in BRIX level, but also showed the undesirabletrait of leaf necrosis. To ensure minimal (or no) introgression ofassociated detrimental traits, experiments were conducted to createrecombinants around the pen1 locus and evaluate these recombinants forboth BRIX levels and necrosis severity. A series of markers wasdeveloped to map the leaf necrosis and BRIX alleles (Table 1). Theoriginal interval was 18.1 cM wide and was narrowed down to a 4 cMregion by evaluating a set of recombinants from a BC1F4 cross betweenthe determinate source LEASE carrying the S. pennellii segment and theindeterminate breeding line FIR-150-2044. After evaluation, it becameclear that this original reduced segment still contained the leafnecrosis. To map the location of the necrosis and BRIX QTLs, anadditional set of SNP markers was developed. In a subsequent mappingusing a BC1F5 population from the FIR-150-2044×LEASE cross, it waspossible to map the leaf necrosis phenotype (at marker loci M1 and M2)at 0.9 cM distance from the BRIX increasing allele, starting at markerlocus M4 (SEQ ID NO:16).

To validate the removal of the deleterious necrosis trait on chromosome1, a second population was developed with a recurrent parent that wasmuch more sensitive to leaf necrosis than the recurrent parent used inthe original mapping. This experiment was run in the winter period toensure optimal conditions for the appearance of leaf necrosis. Sisterlines with and without S. pennellii introgressions were coupled in theexperiment and placed randomly within replications. The plants werephenotyped as previously described and genotyped using the same markersas for the initial mapping population. The results from this experimentconfirmed that a recombination event between marker M2 and markers M3 orM4 would remove the deleterious necrosis trait from the BRIX increasinglocus.

TABLE 1 List of markers and favorable alleles at each marker forbreeding event creation. Ge- SNP netic Public position Posi- Publicposition position Marker in Fav Marker Marker Fav tion marker (bp) SNP(bp) size marker SNP Al- se- Fwd Rev Probe Probe name allele Chr. (cM)(SL3.0) (SL3.0) (bp) (bp) change lele quence primer primer 1 2 M1 S 1154.5 96,680,541-96,680,422 96,680,481 123 61 G/C G 1 2 3 4 5 M2 rp 1155 96,760,850-96,761,549 96,761,293 700 443 T/G T 6 7 8 9 10 M3 donor 1155.7 96,871,011-96,872,011 96,871,421 1001 411 A/G G 11 12 13 14 15 M4donor 1 155.9 96,901,574-96,902,518 96,902,157 972 611 C/G G 16 17 18 1920 M5 donor 1 156.1 96,933,578-96,934,569 96,934,226 995 651 G/A A 21 2223 24 25 M6 donor 1 156.3 97,023,453-97,023,334 97,023,393 121 61 G/A G26 27 28 29 30 M7 donor 1 156.4 96,984,621-96,985,457 96,985,002 843 382G/A G 31 32 33 34 35 M8 donor 1 156.5 96,998,808-96,999,921 96,998,9161114 108 T/A A 36 37 38 39 40 M12 donor 1 156.9 97,061,857-97,062,45797,062,157 601 301 G/A G 56 57 58 59 60 M9 donor 1 15897,236,955-97,237,935 97,237,436 980 481 A/G G 41 42 43 44 45 M13 rp 1158.2 97,261,344-97,261,944 97,261,644 601 301 T/G G 61 62 63 64 65 M10rp 1 161.6 98,155,701-98,155,818 98,155,761 121 61 C/T C 46 47 48 49 50M11 rp 1 164.3 97,847,339-97,845,480 97,845,746 1877 1592 A/G 51 52 5354 55 “rp” = recurrent parent allele “donor” = donor allele

Example 3. Validation of the BRIX Increasing Locus

To ensure that the BRIX increasing locus on chromosome 1 would beefficacious across different genotypic backgrounds, populations of threedifferent tomato types, large medium tomato type (BC₃F₂ population, 96plants), pink tomato type (F2 population, 101 plants) and the cherrytomato type (BC₂F₄ population, 98 plants), segregating for the BRIXallele on chromosome 1 were tested for efficacy of the BRIX allele. Thepopulations were randomly planted in a greenhouse and each plant wasevaluated for genotype and BRIX. In each population three classes ofgenotypes were found: heterozygous, homozygous for the presence of theBRIX allele, and homozygous for the absence of the BRIX allele. The datawas then analyzed for each population independently and it was foundthat the BRIX increasing locus on chromosome 1 had a significant effecton BRIX increase in all three of the populations (Table 2).

TABLE 2 Statistical analysis of three different tomato type populationsfor the effect of the BRIX QTL on chromosome 1. R² QTL Mean BRIX valuePopulation effect P-value −/− +/− +/+ Large medium 0.27 <0.0001 4.845.20 5.46 Pink 0.10 0.0088 6.74 7.24 7.42 Cherry 0.11 0.0042 8.22 8.668.69

Example 4. Breeding Event Creation

To aid breeding efforts, a breeding event donor was developed for theBRIX increasing allele without the detrimental necrosis allele thatcould be used across different breeding programs. The first step increating this event donor was selecting plants with a recombinationevent between M2 (SEQ ID NO:6) and M3 (SEQ ID NO:11) from thefine-mapping population. The plants were then used to develop BC₄F₂material. These plants were then evaluated and compared to the S.lycopersicum parent (FIR-150-244, plants with no necrosis and no BRIXincrease) and the BC₁F₄ plants that contained the full interval (plantswith necrosis and BRIX increase) in a randomized block design with 5replications and 4 plants per plot. Here it was found that all fourtested lines with a recombination event between M2 (SEQ ID NO:6) and M3(SEQ ID NO:11) showed increased BRIX (Table 3) and absence of necrosis(Table 4). These lines could be used as event donors, but one could alsochoose to shorten the amount of S. pennellii DNA at the other side ofthe BRIX allele. To do this one can look for a recombination eventbetween M5 (SEQ ID NO:21) and any marker southwards, or between twomarkers south of M5. For example, between M9 (SEQ ID NO:41) and M10 (SEQID NO:46), which is what was chosen for the breeding event describedhere. Table 1 provides an overview of markers and their preferredalleles for the development of the breeding event described here.Further shortening of the breeding event while maintaining increasedBRIX is possible by selecting for plants with a recombination eventbetween M12 (SEQ ID NO:56) and M13 (SEQ ID NO:61). In this case, oneselects for the S. pennellii donor allele at M12 and the S. lycopersicumrecurrent parent allele at M13.

TABLE 3 Statistical analysis of BRIX measures for breeding event linescompared to the recurrent parent (no BRIX increase) and the BC1F4 plantswith a full interval (BRIX increase) for two harvests. Those materialsnot statistically different from each other are placed in the samegroup. Brix Material Group LSM H1 Group LSM H2 Full interval A 3.9 B 4.8Breeding event A 3.7 A 5.2 FIR-150-2044 B 3.3 C 4.3

TABLE 4 Statistical analysis of leaf necrosis measures for breedingevent lines compared to the recurrent parent (no BRIX increase) and theBC1F4 plants with a full interval (BRIX increase). Those materials notstatistically different from each other are placed in the same group.Necrosis Material Group LSM Check_full interval A 2.9 Breeding event B1.7 RP FIR-150-2044 B 2.0

What is claimed is:
 1. A Solanum lycopersicum plant comprising arecombinant chromosomal segment on chromosome 1, wherein saidchromosomal segment comprises an introgressed BRIX allele from S.pennellii conferring increased BRIX to the fruit of said plant comparedto the fruit of a plant not comprising said allele, and wherein saidchromosomal segment lacks a deleterious allele genetically linked tosaid BRIX allele that confers necrosis to said plant when present. 2.The plant of claim 1, wherein said BRIX allele is located within achromosomal segment flanked in the genome of said plant by: a) markerlocus M3 (SEQ ID NO:11) and marker locus M11 (SEQ ID NO:51) onchromosome 1; b) marker locus M3 (SEQ ID NO:11) and marker locus M9 (SEQID NO:41) on chromosome 1; or c) marker locus M4 (SEQ ID NO:16) andmarker locus M5 (SEQ ID NO:21) on chromosome
 1. 3. The plant of claim 1,wherein said recombinant chromosomal segment comprises a marker locusselected from the group consisting of M1 (SEQ ID NO:1), M2 (SEQ IDNO:6), M3 (SEQ ID NO:11), M4 (SEQ ID NO:16), M5 (SEQ ID NO:21), M6 (SEQID NO:26), M7 (SEQ ID NO:31), M8 (SEQ ID NO:36), M9 (SEQ ID NO:41), M10(SEQ ID NO:46), M11 (SEQ ID NO:51), M12 (SEQ ID NO:56), and M13 (SEQ IDNO:61) on chromosome
 1. 4. The plant of claim 1, wherein said plantcomprises an S. lycopersicum allele at marker locus M2 (SEQ ID NO:6) andan S. pennellii allele at marker locus M3 (SEQ ID NO:11).
 5. The plantof claim 4, further characterized in that said plant comprises an S.pennellii allele at marker locus M9 (SEQ ID NO:41), and an S.lycopersicum allele at marker locus M10 (SEQ ID NO:46).
 6. The plant ofclaim 4, wherein said plant comprises an S. lycopersicum allele at M2(SEQ ID NO:6) and M10 (SEQ ID NO:46) and an S. pennellii allele at amarker locus selected from the group consisting of M3 (SEQ ID NO:11), M4(SEQ ID NO:16), M5 (SEQ ID NO:21), M6 (SEQ ID NO:26), M7 (SEQ ID NO:31),M8 (SEQ ID NO:36), M9 (SEQ ID NO:41), and M12 (SEQ ID NO:56).
 7. Theplant of claim 1, wherein said BRIX allele is located within achromosomal segment in the genome of said plant flanked by 96,871,421 bpand 98,155,761 bp on the Tomato SL3.0 map.
 8. A plant part of the plantof claim
 1. 9. The plant of claim 1, wherein said introgressed BRIXallele is derived from a plant of S. pennellii line LA0716.
 10. Arecombinant DNA segment comprising a BRIX allele that confers increasedBRIX to the fruit of a plant, and lacking a deleterious allelegenetically linked to said BRIX allele that confers necrosis to a plant.11. The recombinant DNA segment of claim 10, wherein said recombinantDNA segment comprises a sequence selected from the group consisting ofM1 (SEQ ID NO:1), M2 (SEQ ID NO:6), M3 (SEQ ID NO:11), M4 (SEQ IDNO:16), M5 (SEQ ID NO:21), M6 (SEQ ID NO:26), M7 (SEQ ID NO:31), M8 (SEQID NO:36), M9 (SEQ ID NO:41), M10 (SEQ ID NO:46), M11 (SEQ ID NO:51),M12 (SEQ ID NO:56), and M13 (SEQ ID NO:61).
 12. The recombinant DNAsegment of claim 10, further defined as comprised within a plant, plantpart, plant cell, or seed.
 13. The recombinant DNA segment of claim 12,wherein said DNA segment confers increased BRIX to the fruit of saidplant.
 14. A method of producing a tomato plant exhibiting fruit withincreased BRIX, comprising: a) crossing the tomato plant of claim 1 withitself or with a second tomato plant of a different genotype to produceone or more progeny plants; and b) selecting a progeny plant comprisingsaid BRIX allele and lacking said deleterious allele.
 15. The method ofclaim 14, wherein selecting said progeny plant comprises detecting an S.Lycopersicum allele at M2 (SEQ ID NO:6) and detecting an S. pennelliiallele at M3 (SEQ ID NO:11).
 16. The method of claim 15, whereinselecting said progeny plant further comprises detecting an S. pennelliiallele at marker locus M9 (SEQ ID NO:41), and detecting an S.lycopersicum allele at marker locus M10 (SEQ ID NO:46).
 17. The methodof claim 14, wherein said progeny plant is an F2-F6 progeny plant. 18.The method of claim 14, wherein producing said progeny plant comprisesbackcrossing.
 19. A method of producing a tomato plant exhibiting fruitwith increased BRIX, comprising introgressing into a plant a BRIX allelewithin a recombinant chromosomal segment flanked in the genome of saidplant by marker locus M3 (SEQ ID NO:11) and marker locus M11 (SEQ IDNO:51) on chromosome 1, wherein said BRIX allele confers increased BRIXto said fruit of said plant compared to a plant not comprising saidallele, and wherein said recombinant chromosomal segment lacks adeleterious allele genetically linked to said BRIX allele that confersincreased necrosis to said plant when present.
 20. The method of claim19, wherein said recombinant chromosomal segment is located within achromosomal segment flanked in said plant by: a) marker locus M3 (SEQ IDNO:11) and marker locus M9 (SEQ ID NO:41) on chromosome 1; or b) markerlocus M4 (SEQ ID NO:16) and marker locus M5 (SEQ ID NO:21) onchromosome
 1. 21. The method of claim 19, wherein said recombinantchromosomal segment comprises a marker locus selected from the groupconsisting of M1 (SEQ ID NO:1), M2 (SEQ ID NO:6), M3 (SEQ ID NO:11), M4(SEQ ID NO:16), M5 (SEQ ID NO:21), M6 (SEQ ID NO:26), M7 (SEQ ID NO:31),M8 (SEQ ID NO:36), M9 (SEQ ID NO:41), M10 (SEQ ID NO:46), M11 (SEQ IDNO:51), M12 (SEQ ID NO:56), and M13 (SEQ ID NO:61).
 22. The method ofclaim 19, wherein said plant comprises an S. lycopersicum allele atmarker locus M2 (SEQ ID NO:6) and an S. pennellii allele at marker locusM3 (SEQ ID NO:11).
 23. The method of claim 22, further characterized inthat said plant comprises an S. pennellii allele at marker locus M9 (SEQID NO:41), and an S. lycopersicum allele at marker locus M10 (SEQ IDNO:46).
 24. The method of claim 19, wherein said introgressing comprisesbackcrossing, marker-assisted selection, or assaying said fruit of saidplant for increased BRIX.
 25. A tomato plant obtainable by the method ofclaim
 19. 26. A method of selecting a tomato plant exhibiting fruit withincreased BRIX, comprising: a) crossing the tomato plant of claim 1 withitself or with a second tomato plant of a different genotype to produceone or more progeny plants; and b) selecting a progeny plant comprisingsaid BRIX allele and lacking said deleterious allele.
 27. The method ofclaim 26, wherein selecting said progeny plant comprises detecting an S.Lycopersicum allele at M2 (SEQ ID NO:6) and detecting an S. pennelliiallele at M3 (SEQ ID NO:11).
 28. The method of claim 27, whereinselecting said progeny plant further comprises detecting an S. pennelliiallele at marker locus M9 (SEQ ID NO:41), and detecting an S.lycopersicum allele at marker locus M10 (SEQ ID NO:46).
 29. The methodof claim 26, wherein said progeny plant is an F2-F6 progeny plant. 30.The method of claim 26, wherein producing said progeny plant comprisesbackcrossing.